From a regulatory standpoint, the principal objective of acute oral toxicity testing is to classify chemicals and formulations according to their intrinsic toxicity, as required for hazard communication and labelling (Council Directive 67/548/EEC and subsequent amendments) [1]. Such classification protects public health by informing safe handling, dosing and use of a substance before it reaches wider clinical application.

Classification is conventionally based on the median lethal dose (LD50) value, defined as the statistically derived single dose of a substance that is expected to cause death in 50% of the animals in an experimental group [1]. The LD50 concept was first introduced by Trevan in 1927 for establishing the toxic potency of biologically active compounds such as digoxin [2]. Since the late 1970s, the classical LD50 test has been criticised on both scientific and animal-welfare grounds [3, 4], prompting successive modifications aimed at reducing animal numbers and suffering while preserving the scientific value of the data [5]. These modifications include the fixed-dose procedure (OECD TG 420) [6], the acute-toxic-class method (OECD TG 423) [7], and the up-and-down procedure (OECD TG 425) [8]. The original LD50 test (OECD TG 401) [9] was formally withdrawn from the OECD test guideline catalogue in 2002.

Polyherbal formulations, though widely perceived as inherently safe because of their traditional use, are not exempt from this requirement — several recent evaluations of similar formulations have shown that systematic acute and sub-chronic toxicity testing remains essential for establishing a credible safety profile before these products can be recommended with confidence [12, 13]. The present study was undertaken to evaluate the acute oral toxicity of the polyherbal formulation FEBRONAL-A Syrup in Wistar rats, following limit-dose testing principles set out in OECD guidelines 420, 423 and 425.

The test formulation was FEBRONAL-A Syrup, a polyherbal formulation manufactured by Unijules Lifesciences, Kalmeshwar, Nagpur (batch no. 0581A170; manufacturing date 01/2017), administered as supplied. Three Wistar rats (two female, one male), 9 weeks old and weighing on average 250 g, were used. The study was approved by the Institutional Animal Ethics Committee of Jawaharlal Nehru Medical College (approval no. JNMC/IAEC/23/12/2016), and all procedures were carried out in accordance with the Committee's guidelines for the humane use of laboratory animals. Animals were acclimatised to their surroundings for 5 days prior to testing, housed separately from stock animals and identified by colour marking. Rats had free access to standard commercial feed, except on the day preceding dosing, and water was provided ad libitum throughout. A single oral dose of the test formulation (2000 mg/kg body weight; 2 ml/animal) was administered by rat feeding needle, consistent with the limit-dose approach common to OECD 420, 423 and 425. Body weight was recorded prior to dosing (Day 1), on Day 7, and prior to sacrifice on Day 14. All procedures were carried out at ambient temperature (27°C). Animals were observed for general appearance and behavioural signs (skin and fur, eyes and mucous membranes, somatomotor activity, tremors/convulsions, salivation, diarrhoea) continuously for the first 6 hours after dosing and thereafter at intervals up to 14 hours, with once-daily observation for mortality and morbidity through Day 14. Body weight was used as a supportive indicator of systemic toxicity, and all animals underwent gross necropsy at study termination. Given the limit-test design and small group size (n = 3) prescribed for acute toxic class/limit-dose testing, data are presented descriptively as individual values and percentage change from baseline; no inferential statistical testing was performed, consistent with the qualitative pass/fail classification objective of the OECD acute toxicity guidelines [7]

1. General appearance and behavioural observations

No treatment-related changes were observed in skin, fur, eyes, mucous membranes, behavioural pattern, salivation or sleep pattern in any animal during the 6-hour and subsequent 14-hour observation windows. No tremors, convulsions, diarrhoea or mortality occurred in any rat, and all three animals survived to the planned termination on Day 14 (Table 1).

Table No. 1:- Clinical Observations of rats at 2,000 mg/kg Dose of FEBRONAL-A SYRUP

Figure 1. Study design and observation timeline.

1. Body Weights

Body weight is a sensitive, non-invasive indicator of systemic toxicity; a treatment-related decline or failure to gain weight is generally considered suggestive of toxicity, whereas normal weight gain supports the absence of systemic effect. All three rats showed a mild, physiological increase in body weight over the 14-day observation period, ranging from 0.0% (Rat 1) to 10.0% (Rat 3) (Table 2, Figures 2 and 3); none of the changes suggested a treatment-related adverse effect.

Table No. 2:- Effect of FEBRONAL-A SYRUP on the body weight of rats at 2000/mg/Kg dose

Figure 2. Body weight trend of individual rats across the observation period.

Figure 3. Percentage change in body weight relative to Day 1.

1. Necropsy

At study termination (Day 14), all animals were euthanised and necropsied; the cranial, thoracic, abdominal and pelvic cavities were opened and examined grossly. No lesions were observed in any organ in any of the three rats (Table 3).

Table 3. Necropsy findings in rats receiving FEBRONAL-A Syrup at 2000 mg/kg.

1. LD₅₀ Value

Since no mortality or overt toxicity was observed at the 2000 mg/kg limit dose, the LD50 of FEBRONAL-A Syrup could not be numerically determined and is reported, per convention, as greater than 2000 mg/kg body weight. Under the Globally Harmonized System (GHS) of Classification and Labelling of Chemicals, a substance with an oral LD50 between 2000 and 5000 mg/kg falls under Category 5 (or is unclassified), representing the lowest acute-toxicity hazard band recognised by the system [11].

In this limit-dose acute oral toxicity study, a single 2000 mg/kg dose of FEBRONAL-A Syrup produced no mortality, no adverse clinical or behavioural signs, no treatment-related change in body weight, and no gross pathological lesion in Wistar rats over 14 days of observation. On this basis, the LD50 of the formulation was estimated to exceed 2000 mg/kg, placing it in GHS Category 5 — the category reserved for substances of low or negligible acute oral toxicity [11]. This finding is consistent with acute toxicity data reported for other polyherbal formulations tested at the same limit dose. Sholikhah et al. found no mortality or toxic signs in Wistar rats receiving a garlic-based polyherbal formulation at 2000 mg/kg, with an estimated LD50 greater than 2000 mg/kg and normal macroscopic and microscopic organ findings on necropsy [12]. Similarly, Niyomchan et al. reported no lethality or toxic signs in rats given the polyherbal formulation NawaTab at doses up to 5000 mg/kg, classifying it under GHS Category 5 as well [13]. The general appearance, behavioural, and necropsy findings in the present study — normal skin, fur, mucous membranes and somatomotor activity, and the absence of gross lesions — mirror the pattern reported in both of these comparator studies, lending external consistency to the present result. The modest, non-progressive gain in body weight observed across all three rats (0–10% over 14 days) is in keeping with normal physiological growth for young Wistar rats of this age and is not indicative of a toxic effect; a treatment-related toxic response would more typically present as weight stagnation or loss rather than the mild gain seen here. The absence of any gross necropsy finding further supports the absence of a treatment-related systemic effect at the tested dose. Several limitations of the present design should be acknowledged when interpreting these results. First, the study used only three animals (two females and one male) rather than a single-sex group of three to five animals as specified by OECD 423 or 425, which normally recommend testing in one sex — usually female, being marginally the more sensitive sex in most acute toxicity studies — to allow the results to be pooled with a starting-dose step in a stepwise class method [7]. The mixed-sex, single-step design used here departs from this formal structure and limits direct comparability with guideline-compliant LD50 classification. Second, the study evaluated only a single fixed dose (2000 mg/kg) without a lower-dose arm or a concurrent vehicle-control group, so it functions as a limit test rather than a full dose-ranging study; this is an accepted approach when there is no expectation of substantial toxicity, but it cannot exclude toxicity at doses that were not tested. Third, the assessment was confined to clinical observation, body weight and gross necropsy; it did not include organ weight measurement, haematological or serum biochemical analysis, or histopathological examination, all of which are recommended in more comprehensive safety evaluations of herbal formulations and were included in both comparator studies discussed above [12, 13]. Fourth, with only three animals, the study is not powered for any inferential statistical comparison, and the reported percentage changes in body weight should be interpreted descriptively rather than as evidence of statistical equivalence to a control group. Taken together, these findings support the preliminary conclusion that FEBRONAL-A Syrup is practically non-toxic on acute oral administration at 2000 mg/kg in rats. However, given the deviations from the full OECD 423/425 protocol noted above, this limit test should be regarded as a preliminary safety signal rather than a definitive hazard classification. A confirmatory study using a single-sex cohort of the recommended group size, together with organ-weight, haematological, biochemical and histopathological endpoints, would strengthen the evidence base, as would sub-chronic (28- to 90-day) repeated-dose toxicity testing to characterise the No Observed Adverse Effect Level (NOAEL) relevant to the intended duration of clinical use, in line with the approach adopted in comparable polyherbal toxicity evaluations [12, 13].

All three animals survived to the end of the study; clinical observation and gross necropsy revealed no treatment-related findings. The LD50 of FEBRONAL-A Syrup was estimated to be greater than 2000 mg/kg body weight, corresponding to GHS Category 5, and the formulation is therefore considered practically non-toxic on single-dose oral administration under the conditions of this limit test. Confirmatory testing with a guideline-compliant single-sex cohort and expanded laboratory and histopathological endpoints is recommended before this finding is generalised to a definitive safety claim.

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